In a chemical method for producing 3-indole-pyruvic acid disclosed by Giovanna De Luca et al., 3-indole-pyruvic acid was produced with a yield of 50 to 62% by reacting tryptophan as a starting material with pyridine aldehyde in the presence of a base for dehydrating a proton acceptor (see JP Sho-62-501912 [Patent Document 1], International Publication 87/00169 Pamphlet [Patent Document 2]). In this method, the required base and pyridine aldehyde are expensive and the yield is low. As a result, production cost are very high. Politi Vincenzo et al. produced 3-indole-pyruvic acid with the yield of 64% by a condensation reaction using indole and ethyl-3-bromopyruvate ester oxime as raw materials followed by acid hydrolysis (see Europe Patent Application Publication No. 421946 [Patent Document 3]). This method, however, requires a purification step using silica gel, the yield is low, the raw materials are expensive and the industrial production cost is very high.
On the other hand, an enzymatic method for production of 3-indole-pyruvic acid using an aminotransferase is known (see the following reaction formula 1).

In an example of this method, 3-indole-pyruvic acid is generated from 40 mM L-tryptophan (L-Trp) and 80 mM 2-ketoglutaric acid by reacting L-Trp and 2-ketoglutaric acid with an L-tryptophan aminotransferase derived from Candida maltosa and purifying the resulting 3-indole-pyruvic acid using an ion exchange resin to achieve the yield of 721 (see Bobe Ruediger et al., East Germany Patent DD 297190 [Patent Document 4]). By reacting L-Trp and 2-ketoglutaric acid with an aspartate aminotransferase to generate 3-indole-pyruvic acid, which is then purified by extracting the reaction solution with petroleum ether and fractionating by column chromatography and the fraction is collected (see Mario Materazzi et al., JP Sho-59-95894-A [Patent Document 5]). Aminotransferases encoded by an aspC gene and a tyrB gene derived from Escherichia coli is described in International Publication No. 2003/091396 Pamphlet [Patent Document 6] and US Patent Application Publication No. 2005/0282260 [Patent Document 7]. In these methods of using an aminotransferase, the yield is low and a keto acid (e.g. 2-ketoglutaric acid) is required as an amino group acceptor in addition to L-Trp. In addition, an amino acid corresponding to the amino group acceptor present in an amount of equivalent moles to 3-indole-pyruvic acid to be generated is produced as a byproduct. Furthermore, an excessive amount of keto acid relative to L-Trp is added to the reaction system in order to enhance the yield. Thus, unreacted keto acid remains after the reaction. Due to these reasons, a purification step employing an ion exchange resin is required in order to collect the 3-indole-pyruvic acid from the reaction solution. Thus, manipulation of the reaction is complicated and the cost is high.
Another known method for producing 3-indole-pyruvic acid from L-Trp employs an L-amino acid oxidase a. In this regard, however, 3-indole-pyruvic acid is decomposed into indoleacetic acid (see the following reaction formula 3) by hydrogen peroxide which is produced as a byproduct when tryptophan is oxidized by L-amino acid oxidase (see the following reaction formula 2). Thus, a method of decomposing hydrogen peroxide by adding catalase to the reaction system (see the following reaction formula 4) is proposed (see U.S. Pat. No. 5,002,963 to De Luca, et al., 1991 [Patent Document 8]).



In the above method, an immobilized enzyme column is used in which L-amino acid oxidase derived from a snake venom and catalase derived from bovine liver have been immobilized to a carrier. Then, a solution containing L-Trp is passed through the column to react, and the produced 3-indole-pyruvic acid is adsorbed to an ion exchange column, eluted with methanol, subsequently exsiccated and collected. However, in this method, only 0.2 g of 3-indole-pyruvic acid is acquired from 0.5 g of L-Trp, and the yield is low (40%). Further, in this method, the steps such as immobilizing the enzymes and purifying by the ion exchange resin are complicated and it is also necessary to collect or recycle unreacted L-Trp, which leads to an increased cost.
The L-amino acid oxidase derived from microorganisms is provided by John A. Duerre et al., who crudely purified L-amino acid oxidase (deaminase) derived from Proteus rettgeri and detected an oxidation activity for L-Trp by an activity measurement method of detecting an amount of consumed oxygen (see Journal of Bacteriology, 1975, vol. 121, No. 2, p656-663 [Nonpatent Document 1]). Furuyama et al. confirmed that L-phenylalanine oxidase derived from Pseudomonas sp.P-501 also acted upon L-Trp by an activity measurement method by detecting the amount of consumed oxygen (see Noda Institute for Scientific Research, JP Sho-57-146573-A [Patent Document 9]).
However, in any of these methods, the oxidase activity was detected by measuring the amount of consumed tryptophan, the amount of consumed oxygen or the amount of produced hydrogen peroxide. The indole-pyruvic acid was not directly quantified. This seems to be because 3-indole-pyruvic acid is decomposed into indoleacetic acid by hydrogen peroxide produced by the reaction with amino acid oxidase. On the other hand, there is no example in which 3-indole-pyruvic acid is generated using a microbial cell or a treated microbial cell. Further, how tryptophan is metabolized by the microorganism and what metabolite is generated by the microorganism are unknown.
The microorganisms having oxidase activity and belonging to genera Achromobacter, Proteus, Morganella, Pseudomonas and Neurospora are disclosed in International Publication No. 03/056026 Pamphlet (patent Document 10). However, when 3-indole-pyruvic acid was industrially produced on a large scale, there was a limit to produce it by a microbial cell reaction alone.
Further, in the method of using aminotransferase or the method of using L-amino acid oxidase derived from the snake venom among the aforementioned technology known publicly, the reaction yield is low, keto acid as the byproduct or unreacted L-tryptophan remains and is mixed in the reaction solution. Thus, a chromatographic separation step is required to collect the 3-indole-pyruvic acid, thus the manipulation is complicated and the cost is high.
Under the circumstance as above, it is required to develop the method for producing 3-indole-pyruvic acid inexpensively and conveniently.    Patent Document 1: JP Sho-62-501912-A    Patent Document 2: International Publication WO87/00169 Pamphlet    Patent Document 3: Europe Patent Application Publication No. 421946    Patent Document 4: East Germany Patent DD 297190    Patent Document 5: JP Sho-59-95894-A    Patent Document 6: International Publication No. WO2003/091396    Pamphlet    Patent Document 7: US Patent Application Publication No. 2005/0282260    Patent Document 8: U.S. Pat. No. 5,002,963    Patent Document 9: JP Sho-57-146573-A    Patent Document 10: International Publication No. 03/056026 Pamphlet    Nonpatent Document 1: Journal of Bacteriology, 1975, vol. 121, No. 2, p656-663.